Infarct-sparing effect of myocardial postconditioning is dependent on protein kinase C signalling

Transcription

1 Cardiovascular Research 70 (2006) Abstract Infarct-sparing effect of myocardial postconditioning is dependent on protein kinase C signalling Amanda J. Zatta a, *, Hajime Kin a, George Lee a, Ningping Wang a, Rong Jiang a, Robert Lust b, James G. Reeves a, James Mykytenko a, Robert A. Guyton a, Zhi-Qing Zhao a, Jakob Vinten-Johansen a Department of Cardiothoracic Surgery, Carlyle Fraser Heart Centre/Crawford Long Hospital, Emory University School of Medicine, Atlanta, GA , United States b Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC , United States Received 6 September 2005; received in revised form 12 November 2005; accepted 29 November 2005 Available online 27 January 2006 Time for primary review 25 days Objective: Using non-selective and selective protein kinase C (PKC) ( and y isoform inhibitors, we tested the hypothesis that the cardioprotective phenotype invoked by postconditioning (postcon) is dependent on PKC signalling. Furthermore, we determined whether postconditioning alters ppkc( and/or ppkcy in cytosolic and mitochondrial fractions. Methods: Male Sprague Dawley rats underwent 30 min left coronary artery (LCA) occlusion followed by 3 h of reperfusion. Rats were randomised to the following groups: Untreated, no intervention either before or after LCA occlusion; Postcon, 3 cycles of 10-s full reperfusion and 10-s re-occlusion, initiated immediately at the onset of reperfusion; Chelerythrine (non-selective PKC inhibitor, 5 mg/kg) T postcon; Rottlerin (PKCy inhibitor, 0.3 mg/kg) T postcon; KIE1-1 (PKC( inhibitor, 3.8 mg/kg) T postcon. A subset of rats was employed to assess ppkc( and/or ppkcy in sham, Isch/RP (30-min LCA occlusion followed by 30-min reperfusion), and postcon-treated hearts. Results: Infarct size, expressed as area of necrosis as a percentage of the area at risk, AN/AAR (%), was significantly reduced by postcon compared to control (untreated) rats (39T2% vs. 53T1% in control, P <0.001). Treatment with chelerythrine alone or the PKC( antagonist KIE1-1 alone at reperfusion had no effect on infarct size compared to control. In contrast, the infarct-sparing effect of postcon was abrogated by non-selective PKC inhibition and PKC( antagonism (50T2% and 50 T1%, respectively, P <0.002). Inhibition of PKCy reduced infarct size to values comparable to that in postcon group (36 T3% vs. 39T2%). However, postcon in the presence of PKCy inhibitor did not enhance the infarct-sparing effects (38 T 2%). In addition, ppkc( in postcon hearts was significantly higher in the total cell homogenate (10338 T 1627 vs T 608 in Isch/RP, arbitrary units), and ppkcy translocation to mitochondria was significantly less (>2-fold decrease) compared to Isch/RP. Conclusion: These data suggest that postcon modulates PKC during early reperfusion by increasing PKC( expression and translocation to a site other than the outer mitochondrial membrane, and limits translocation of PKCy to mitochondria and associated deleterious signalling. D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. Keywords: Postconditioning; Myocardial infarction; Mitochondria; Protein kinase C; Protein phosphorylation; Reperfusion injury 1. Introduction * Corresponding author. Cardiothoracic Research Laboratory, Crawford Long Hospital 550 Peachtree Street, NE, Atlanta, GA , United States. Tel.: ; fax: address: azatta@emory.edu (A.J. Zatta). Postconditioning (postcon) is a novel strategy to harness nature s protection against myocardial ischaemia reperfusion injury. Postconditioning is induced by brief repetitive mechanical interruptions of the early moments of reperfusion (i.e. a specified algorithm). The cardioprotective /$ - see front matter D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. doi: /j.cardiores

2 316 A.J. Zatta et al. / Cardiovascular Research 70 (2006) phenotype invoked by postconditioning has been demonstrated to involve a reduction in infarct size, apoptosis as well as vascular injury. These physiological manifestations have been associated with a myriad of cellular and subcellular effects including attenuated generation of reactive oxygen species (ROS) and oxidant-mediated injury, endogenous activation of adenosine receptors, activation of K ATP channels, decreased intracellular and mitochondrial calcium accumulation and closure of the mitochondrial permeability transition pore (mptp) (see review [1]). Indeed, postconditioning sets in motion triggers (adenosine, nitric oxide) and molecular signals (survival kinases and anti-apoptotic enzymes) during early reperfusion that are functionally linked to reduced cell death [1 5], provoking renewed interest in reperfusion injury, the molecular signals involved, and therapeutic strategies for treatment. One family of signalling proteins commonly linked to the modulation of ischaemia reperfusion injury is protein kinase C (PKC), in particular the PKC isozymes PKC( and PKCy. Importantly, PKCy has been implicated as a key signalling element in myocardial and cerebral reperfusion injury processes [6 8]. PKCy has been demonstrated to rapidly translocate to mitochondria within the first 5 min of reperfusion [9], and is associated with increased superoxide anion (O 2 ) generation, loss in mitochondrial function, as well as enhanced release of cytochrome c and downstream pro-apoptotic factors [9,10]. Conversely, translocation of PKC( has been determined to be pivotal in triggering cardioprotective effects of ischaemic and anaesthetic preconditioning [11 16]. Curiously, recent evidence indicates that volatile anaesthetics administered at reperfusion can also mimic postconditioning [17]. In addition, PKC( has been reported to exert cardioprotection by inhibiting the mptp [18], which is also purportedly inhibited by postconditioning [19]. Accordingly, we postulated that postconditioning may trigger the activation of PKC( and/ or limit PKCy translocation, ultimately leading to a reduction in infarct size. 2. Materials and methods Investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publications No , revised 1996) In vivo rat surgical preparation One hundred and thirty male Sprague Dawley rats weighing g were initially anaesthetised via intraperitoneal administration of sodium pentobarbital (40 mg kg 1 ). A tracheotomy was performed, and the trachea was intubated with a cannula connected to a rodent ventilator (Harvard Rodent Ventilator Model 683, Holliston, MA). Rats were ventilated with O 2 -enriched room air at breaths per minute, with tidal volumes set to 1.0 ml/100 mg body weight. Arterial po 2, pco 2 and ph were monitored (Stat Profile M, Nova Biomedical, Waltham, MA) during the experiment and values were maintained within a normal physiological range (ph ; pco 2, mm Hg; and po 2, mm Hg) by adjusting the ventilatory rate and tidal volume, or by intravenous administration of sodium bicarbonate. Body temperature was kept at a constant 37 -C by using an adjustable heating pad. The left carotid artery was cannulated with a 24-gauge angiocatheter connected to a fluid-filled pressure transducer to continuously monitor phasic and mean arterial pressure (MAP) and heart rate (HR) using the EMKA data acquisition and analysis system (EMKA Technologies Inc., Falls Church, VA). The right external jugular vein was cannulated for delivery of anaesthesia and drug treatments. The chest was opened via a left thoracotomy through the fourth or fifth intercostal space, and the ribs were gently retracted to expose the heart. After pericardiotomy, a 6-0 proline (Ethicon, NJ) ligature was placed under the left coronary artery (LCA), and the ends of the suture were threaded through polyethylene tubing (PE-50) to form a snare for reversible LCA occlusion. Following surgical preparation (prior to LCA occlusion), a bolus dose of sodium heparin (100 U/kg) was administered to ensure heparinisation throughout the experiment. The LCA was occluded by tightening the snare using a lightweight hemostatic clamp. Ischaemia was confirmed by a transient decrease in blood pressure and cyanosis on the myocardial surface. Reperfusion was indicated by an epicardial hyperaemic response and rapid disappearance of cyanosis Experimental protocol for in vivo infarct size studies Following stabilisation of haemodynamics and blood gases, rats were subjected to a 30-min LCA occlusion (index ischaemia) followed by 3 h of reperfusion with or without a postconditioning stimulus (3 cycles of 10-s reperfusion and 10-s re-occlusion) initiated at the onset of reperfusion. In appropriate groups, antagonists were administered as a slow infusion over a 5-min period starting 5 min prior to reperfusion. Rats were randomised to one of nine groups (Fig. 1): (1) Untreated (Control), no intervention either before or after LCA occlusion (n =11). (2) Vehicle control, dimethyl sulfoxide (DMSO, <300 Al/kg) alone (n =8). (3) Postcon, initiated immediately at the onset of reperfusion, 3 cycles of 10 s full reperfusion and 10 s reocclusion (total intervention time of 1 min) (n = 12). (4) Chelerythrine (non-selective PKC inhibitor, 5 mg/kg) alone (n = 10). (5) Chelerythrine + Postcon, postconditioning stimulus initiated in the presence of antagonist (n = 8). (6) Rottlerin (PKCy inhibitor, 0.3 mg/kg) alone (n = 10). (7) Rottlerin+Postcon (n =9). (8) KIE1-1 (Tat conjugated PKC( inhibitor, 3.8 mg/kg; Tat is a cell-permeating carrier peptide) alone (n =4). (9) KIE1-1+Postcon (n =8).

3 A.J. Zatta et al. / Cardiovascular Research 70 (2006) Fig. 1. Experimental protocol employed to investigate if the infarct-sparing effect postconditioning is dependent on PKC signalling. In preliminary studies, the efficacy of the Tat conjugated PKC( inhibitor, KIE1-1, was verified by administering the peptide in the absence and presence of preconditioning (precon). As mentioned previously, the cardioprotective effects of preconditioning involve the activation of PKC( [11 16]. To test KIEI-1 in precon, rats were assigned to one of the following groups: Precon, 5-min LCA occlusion followed by 10 min of reperfusion before index ischaemic event (n = 10); Tat carrier control (cell-permeating carrier peptide, 3.8 mg/kg) + Precon, infused over a 5-min period starting 5 min prior to the precon stimulus (n =4); KIE1-1 alone (3.8 mg/kg), infused over a 5-min period starting 20 min prior to index ischaemia (n = 4); KIE1-1 + Precon (n =8) Determination of infarct size Upon completion of the experiment, the LCA was reoccluded and the AAR was delineated by injecting 1.0 ml of 20% Unisperse blue dye via the external jugular vein. The heart was rapidly excised and placed into 0.9% saline. An individual blinded to the protocol subsequently separated the LV from remaining tissue and thinly ( 2 mm) crosssectioned the LV before separating the AAR (unstained) from blue stained non-ischaemic zone. The AAR was incubated for 10 min in a phosphate-buffered 1% 2,3,5- triphenyltetrazolium chloride (TTC) solution at 37 -C, enabling a clear differentiation of necrotic tissue (area of necrosis, AN). AN and AAR were determined by gravimetric analysis. Infarct size was expressed as a percentage of the AAR (AN/AAR) Isolation of mitochondria from rat heart A subset of rats was randomly assigned to one of three groups: (1) Sham, subjected to surgical manipulation without LCA occlusion (n =3); (2) Isch/RP, 30-min LCA occlusion and 30-min reperfusion (n = 6); (3) Postcon, postconditioning stimulus (3 cycles of 10-s reperfusion and 10-s re-occlusion) initiated at the onset of reperfusion following index ischaemia (n = 6). Cytosolic and mitochondrial fractions were isolated by differential centrifugation as described by Sayen et al. [20]. Preliminary Western blot studies (data not shown) determined that our mitochondrial isolation process resulted in <6% sarcolemma contamination and negligible sarcoplasmic reticulum contamination (contamination in mitochondria was calculated as a percentage of total cell lysate). Briefly, the AAR was delineated with blue dye at the end of the experimental protocol, the hearts were rapidly excised and placed into ice cold MSE buffer [in mmol/l: 220 mannitol, 70 sucrose, 2 EGTA, 5 MOPS (ph 7.4), 2 taurine supplemented with 0.1% fatty acid-free bovine serum albumin (BSA) and protease inhibitor cocktail]. Following separation of the AAR from the non-ischaemic zone, 200 mg of minced AAR tissue was homogenised in 3 ml of MSE buffer with a Polytron-type tissue grinder at 11,000 RPM for 2.5 s, followed by 2 quick strokes at 500 RPM with a loose fitting Potter-Elvehjem tissue grinder. The homogenate was then transferred to a 15-ml conical polypropylene tube and centrifuged twice at 600g for 5 min each (4 -C). The pellet was discarded and the supernatant was then centrifuged for 10 min at 3000g (4 -C) to pellet

4 318 A.J. Zatta et al. / Cardiovascular Research 70 (2006) mitochondria. The supernatant (crude cytosol) was collected and further purified by centrifugation at 100,000g for 1 h, at 4-C. The mitochondrial pellet was then rinsed and gently re-suspended in 1 ml MSE buffer before repeating the 3000g spin for 10 min. The final mitochondrial pellet was rinsed and re-suspended in 200 Al of MSE buffer. Cytosolic and mitochondrial aliquots were frozen until Western blot analysis was performed. Protein concentration was determined using the Bradford assay, and for all experiments, an equal amount of mitochondrial and cytosolic protein was loaded on gels Western immunoblotting Translocation ppkcy and ppkc( levels were assessed using 60 Ag of mitochondrial and cytosolic protein resolved on 8% SDSpolyacrylamide gels and transferred onto nitrocellulose membranes (Hybond ECL; Amersham, Piscataway, NJ). After transfer, the membranes were washed 3 in TBS-T (0.1% Tween-20) and then blocked for 1 h at room temperature with 5% milk in TBS-T with 0.1% azide. Membranes were then probed with the following primary antibodies: ppkcy (Ser 643) polyclonal rabbit antibody (1:1000 in 5% BSA in TBS-T, Cell Signalling Technology), ppkc( (Ser 729) polyclonal rabbit antibody (1:1000 in 3% milk in TBS-T, Upstate) or tpkc( polyclonal rabbit antibody (1:1000 in 3% milk in TBS-T, Upstate). Antiporin (anti-vdac) polyclonal rabbit antibody (1:2000 in 3% milk in TBS-T, Calbiochem) and anti-actin monoclonal mouse antibody (1:5000 in 3% milk in TBS-T, Sigma) were used to determine equal loading and to verify fraction purity (mitochondrial and cytosolic, respectively). Membranes were subsequently washed four times in TBS-T and then subjected to the appropriate horseradish peroxidase conjugated secondary antibody for 1 h at room temperature. Protein bands were visualised using a chemiluminescence system (ECL kit; Amersham, Piscataway, NJ) and densitometry was quantitated using NIH ImageJ software. Individual ppkc isoform densities were expressed as a ratio of their respective actin or VDAC control and multiplied by 100. To accurately measure translocation, cytosolic and mitochondrial fractions from the same heart were run on the same gel. Translocation was calculated by dividing the normalized mitochondria density by the normalized cytosolic density and multiplying by 100 (mito/cyto ratio) Detection of ppkce in total cell homogenate Additional rat experiments (same protocol as described in Section 2.4) were undertaken to extract total proteins from the AAR tissue: Sham (n = 1), Isch/R (n = 4) and Postcon (n =4). 100 mg heart tissue was placed in 500 Al1 lysis buffer (Cell Signalling; Beverly, MA) with added PMSF (1 mm), and homogenised at 4 -C for 20 s. Samples were subsequently incubated on wet ice for 30 min and then centrifuged at 16,000g for 10 min at 4 -C. Aliquots were stored at 80 -C until Western blot analysis was performed. Following protein concentration determination using the Bradford assay, 50 Ag of total protein was resolved on 8% SDS-polyacrylamide gel and standard Western blot procedures were carried out as described above Chemicals Rottlerin was purchased from BIOMOL International (Plymouth Meeting, PA) and the PKC( inhibitor, KIE1-1 was kindly supplied by KAI Pharmaceuticals (South San Francisco, CA). Protease inhibitor cocktail was obtained from Roche Applied Science (Indianapolis, IN) and MOPS buffer from Mediatech (Herndon, VA). All other drugs were purchased from Sigma/RBI (St. Louis, MO). Chelerythrine and KIE1-1 were dissolved directly in 0.9% saline, whereas Rottlerin was dissolved in <300 Al/kg DMSO. Previous studies from our lab have shown that this concentration of DMSO has no effect on haemodynamics or infarct size in the rat model [21], confirmed by results below. Chelerythrine was employed as it acts as a selective PKC antagonist, effectively targeting the catalytic site of all PKC isoforms [22]. We used chelerythrine at a dose (5 mg/kg) that has previously been demonstrated to inhibit the translocation of PKC( [15] and PKCy [23] and associated cardioprotection [15,23,24]. We additionally used the selective PKCy inhibitor, rottlerin (0.3 mg/kg) which has been reported to prevent PKCy translocation in preconditioned animals [25] and the highly selective PKC( antagonist, KIE1-1 (3.8 mg/kg) (C-EAVSLKPT) [26] which was conjugated to the cell permeable peptide, Tat (C-YGRKKRRQRRR) to enhance cellular uptake [27] Statistical analysis All values are reported as mean T S.E.M. Data were analysed using SigmaStat 2.0 for Windows statistical software package (SPSS, Inc., Chicago, IL). One-way analysis of variance (ANOVA) with a Student Newman Keuls post-hoc test was employed to determine if any significant differences existed between groups for individual parameters. Statistical analysis of infarct size was performed over all groups simultaneously; data may be regrouped for a more logical presentation. In all tests, P <0.05 was considered indicative of statistical significance. 3. Results 3.1. Haemodynamic data The haemodynamic data are summarised in Table 1. There were no significant differences observed between control and treatment groups for any variable [HR, MAP or

5 A.J. Zatta et al. / Cardiovascular Research 70 (2006) Table 1 Haemodynamic variables during the course of the experiment Group Baseline 25-min ischaemia 180-min reperfusion Heart rate MAP RPP Heart rate MAP RPP Heart rate MAP RPP Control 368T13 91T3 33T2 361T11 85 T5 31T2 306T10 91T3 28T1 Postcon 348T12 88T3 32T2 338T10 93T4 34T2 303T7 94T3 30T1 Chelerythrine 356T25 82T7 30T3 334T22 92T8 31T3 284T18 91T7 26T2 Postcon+Chelerythrine 347T11 92 T3 32T2 328T9 90T4 30T2 302T12 93T2 28T1 Vehicle 381T16 83T6 32T4 379T12 82T9 31T4 323T26 81T9 27T4 Rottlerin 389T9 88T4 34T2 400T11 105T5 42T3 353T15 88T4 31T2 Postcon+Rottlerin 402T9 96T6 39T2 414T11 94 T5 39T3 394T16T 88T4 34T1 KIE T14 104T14 43T7 345T12 94T4 32T2 342T34 89T6 31T5 Postcon+KIE T13 82T3 30T2 393T18 90T4 36T3 371T15T 91T7 34T3 MAP, mean-arterial pressure; RPP, rate-pressure product/1000. * P <0.05 vs. values in control group. rate-pressure product (RRP)] at baseline or at the end of ischaemia. Significant differences in HR were observed at the end of reperfusion between control and postcon hearts treated with rottlerin or KIE1-1. Significant differences in HR were also detected between control (306T10 bpm) and hearts subjected to precon (383T12), precon +Tat carrier control (381 T11), KIE1-1 (preisch) (390 T 19) and precon + KIE1-1 (394 T 12) ( P < 0.05). No significant differences in MAP or RPP were observed at the end of reperfusion Efficacy of the Tat conjugated PKCe inhibitor, KIE1-1 on preconditioning Preliminary studies revealed that the Tat conjugated PKC( inhibitor, KIE1-1, at a concentration of 3.8 mg/kg, had no effect on infarct size when administered as a pretreatment alone (51T1% vs. 53T1% in control), however when given as a pretreatment before the precon stimulus it abolished the infarct size reduction with precon (44T2% vs. 34T2% in precon, P <0.007). Importantly, the Tat carrier control (peptide vehicle) did not have an effect on precon cardioprotection (35T1% vs. 34 T2% in precon) Infarct size The area placed at risk by LCA occlusion, expressed as a percent of left ventricular mass (AAR/LV), was comparable among groups ( 38 42%) (Table 2). Infarct size, expressed as a percentage of area at risk (AN/AAR), was significantly reduced in the postcon group (39T2%) compared with the control or vehicle rats (53 T 1% or 54T3%, respectively, P <0.001) (Table 2, Fig. 2). Reperfusion treatment with chelerythrine (non-selective PKC inhibitor, 5 mg/kg) and KIE1-1 (selective PKC( antagonist, 3.8 mg/kg) alone had no effect on infarct size compared to control (Table 2, Fig. 2A). However, chelerythrine and the PKC( inhibitor KIE1-1 abrogated postcon infarct size reduction (50 T 2% and 50 T 1%, respectively, P < 0.002) (Table 2, Fig. 2A). Interestingly, rottlerin (selective PKCy blocker, 0.3 mg/kg) alone resulted in similar protection as observed in postcon hearts (36T3% vs. 39T2% in postcon) (Table 2, Fig. 2B). However, the infarct-sparing effect of postcon was not enhanced by rottlerin (38 T 2%), suggesting that postcon may involve modulation of PKCy during early reperfusion (Table 2, Fig. 2B) Western blot analysis In order to investigate the significance of the molecular pathways inferred by the infarct size data, western blot analysis was performed on total cell homogenate, cytosolic and mitochondrial fractions obtained from sham, Isch/RP and postcon hearts harvested at 30-min reperfusion. Compared to Isch/RP (4165 T 608, arbitrary units), postcon resulted in significantly higher total ppkc( levels, similar to sham (10,338 T 1627 vs. 10,044 in sham, arbitrary units) (Fig. 3). However, despite the observation that postcon resulted in significantly higher ppkc( in the total cell homogenate (Fig. 3), we did not detect any significant differences in ppkc( between groups in the cytosolic or Table 2 Infarct size data Group n Body weight (g) LV weight (mg) AAR/LV (%) AN/AAR (%) Control T13 587T26 39T2 53T1. Postcon T14 588T30 41T3 39T2* Chelerythrine T7 620T15 40T3 48T1. Postcon+Chelerythrine 8 327T8 661T11 42 T3 50T2. Vehicle 8 369T6 698T15 38T3 54T3. Rottlerin T5 685T13 42T2 36T3* Postcon+Rottlerin 9 339T9 642T20 40T1 38T2* KIE T6 738T46 39T1 50T1. Postcon+KIE T7 664T19 40T2 50T1. Values are expressed as meants.e.m. All treatment groups were assessed simultaneously using one-way ANOVA for individual parameters. * P <0.05 vs. values in control and vehicle group.. P <0.05 vs. values in post-con group.

6 320 A.J. Zatta et al. / Cardiovascular Research 70 (2006) Fig. 2. Effect of selective PKC isoform blockade on postconditioninginduced infarct size reduction following 30-min occlusion and 180-min reperfusion. (A) Effects of non-selective PKC, chelerythrine (5 mg/kg) or PKC( inhibitor, KIE1-1 (3.8 mg/kg) in the absence or presence of postcon. (B) Effects of PKCy inhibitor, rottlerin (0.3 mg/kg) in the absence or presence of postcon. All values are meansts.e.m. *P <0.05 vs. control group;.p <0.05 vs. postcon group. Fig. 3. ppkc( expression is significantly higher in total cell homogenate following postconditioning. *P <0.05 vs. sham;.p <0.05 vs. Isch/RP group. Fig. 4. PKC( does not translocate to mitochondria in response to postconditioning. (A) Western blot analysis of cytosolic (cyto) and mitochondrial (mito) fractions (60 Ag) for ppkc(, tpkc(, VDAC and actin. (B) ppkc( levels in cytosolic and mitochondrial fraction normalized to their loading control, actin and VDAC, respectively. (C) ppkc( translocation (% ratio of mito/cyto fraction). All values are meansts.e.m.; n =3 sham, n =6 Isch/RP, n =6 postcon. *P <0.05 vs. sham;.p <0.05 vs. Isch/RP group. mitochondrial fractions (Fig. 4B and C). Although ppkc( was present at very low levels, tpkc( was detected in all groups including sham, consistent with localisation on the outer membrane of the mitochondria. Importantly, no differences in tpkc( were observed between sham, Isch/ RP or postcon in the cytosolic and mitochondrial fractions. With regard to PKCy, there was a trend for ppkcy levels in Isch/RP to decrease in the cytosolic fraction (72T9 vs. 101T 2 in sham) while being significantly increased in the mitochondrial fraction (23T4 vs. 7T1 in sham, P <0.03) during early reperfusion (Fig. 5B). Moreover, when expressed as a percent translocation between the mitochondrial and cytosolic fractions (mito/cyto), Isch/RP was associated with a 5-fold increase in ppkcy translocation to the mitochondria relative to sham (35T8 vs. 7T1 in sham, P < 0.04) (Fig. 5C). Importantly, our data revealed that postcon hearts exhibited significantly less ppkcy translocation to mitochondria (> 2-fold decrease) relative to hearts

7 A.J. Zatta et al. / Cardiovascular Research 70 (2006) Fig. 5. Postconditioning limits translocation of PKCy to mitochondria. (A) Western blot analysis of cytosolic (cyto) and mitochondrial (mito) fractions (60 Ag) for ppkcy, VDAC and actin. (B) ppkcy levels in cytosolic and mitochondrial fraction normalized to their loading control, actin and VDAC, respectively. (C) ppkcy translocation (% ratio of mito/cyto fraction). All values are meansts.e.m.; n =3 sham, n =6 Isch/RP, n =5 postcon. *P <0.05 vs. sham;.p <0.05 vs. Isch/RP group. subjected to Isch/RP alone (16 T 2 vs. 35 T 8 in Isch/RP, P <0.05) (Fig. 5C). 4. Discussion The results of the current investigation suggest that the cardioprotective effects of postconditioning are dependent on PKC signalling. Our data demonstrate that the infarctsparing effects of postconditioning are reversed by selective PKC( inhibition, suggesting that postconditioning may be triggered by endogenous activation of PKC(. This finding was also corroborated by our Western blot data, which demonstrate that postconditioning is associated with significantly higher levels of ppkc( in total cell homogenate relative to the untreated Isch/RP group, an effect that appeared independent of its translocation to mitochondria. In addition, our data support previous observations that reperfusion stimulates PKCy translocation to mitochondria and that inhibition of PKCy during reperfusion substantially reduces infarct size [9,28]. Importantly, we extend these findings by revealing that the cardioprotective effects of postconditioning are not enhanced by PKCy inhibition, and that postconditioning prevents significant reperfusion-induced PKCy translocation to mitochondria. Taken together, these data suggest that postconditioning modulates PKC during early reperfusion by increasing PKC( expression and translocation to a site other than the outer mitochondrial membrane, and limits translocation of PKCy to mitochondria and associated deleterious signalling. The concept of a signalling role for PKC during reperfusion is consistent with studies showing that endogenous activators of PKC such as adenosine [21] and opioids [29] are involved in the cardioprotective effects of postconditioning Evidence for a role of PKCe activation in postconditioning cardioprotection PKC( is an essential component involved in the triggering process of ischaemic preconditioning [11,14,15,30 34]. Importantly, PKC( is activated not only by a preconditioning stimulus, but also by treatments that mimic preconditioning such as adenosine [35], nitric oxide (NO ) [36], volatile anaesthetics [12,13,16,37], and exogenously applied PKC( agonists [38]. Interestingly, many of these same triggers (adenosine, NO ) and signals (survival kinases and anti-apoptotic enzymes) that are implicated in preconditioning and the activation of PKC( have also been identified to play a role in postconditioning [2 5,19,21,39]. A recent example is the finding that isoflurane, a well-known anaesthetic preconditioning agent that mediates cardioprotection via activation of PKC( [12,40,41], also invokes a postconditioning-like effect when administered at the onset of reperfusion [17]. Therefore, we hypothesise that PKC( may also play a role in postconditioning-mediated protection. In the present study, we demonstrated that selective PKC( inhibition (at a concentration that inhibited preconditioning) reversed the infarct-sparing effects of postconditioning, thus providing functional evidence for the involvement of endogenously activated PKC( in the modification of reperfusion injury processes. Only one other study has examined the effects of PKC( activation on reperfusion injury. Inagaki et al. demonstrated in Langendorff perfused rat hearts that exogenous activation of PKC( at the onset of reperfusion did not improve contractile function or reduce creatine kinase (CK) activity [6]. The major difference between this and the current study, apart from the end points measured and the model employed, is the use of exogenous versus endogenous activation of PKC( to modify reperfusion injury processes. Indeed, it is entirely possible that endogenous activation of PKC( is functionally involved in cardioprotection, and that exogenously applied PKC(

8 322 A.J. Zatta et al. / Cardiovascular Research 70 (2006) activators are not as effective in eliciting the same degree of protection. Additional studies are needed to reconcile the importance of both endogenous and exogenous PKC( specifically during reperfusion. Since cardioprotection mediated by activation of PKC( has been reported to redistribute to mitochondria with ischaemic and pharmacological preconditioning [15,42] where it is thought to orchestrate protection, we attempted to elucidate if postconditioning triggers PKC( translocation to mitochondria. Indeed, Baines et al. have shown that cardiac mitochondria constitutively express PKC( where it can interact with (1) MAPK to form protective signalling modules [43] and (2) mptp to inhibit pore opening in the heart [18]. As mentioned previously, accumulating evidence suggest that the postconditioning cardioprotection involves inhibition of mptp opening [19,44]. However, while our data show that PKC( is localised on the outer membrane of mitochondria, and that postconditioning resulted in significantly higher ppkc( expression in total cell homogenate compared to Isch/RP, we did not detect any differences in ppkc( in the cytosolic or mitochondrial fraction. There are several explanations why we did not observe any translocation of ppkc( to mitochondria with postconditioning. Firstly, it is possible that PKC( translocates to the inner mitochondrial membrane to activate protective downstream signalling, rather than translocating to the outer mitochondrial membrane. Indeed, very recent evidence by Costa et al. indicates that PKC( plays a critical role in transmitting a cardioprotective signal from activated PKG (located on the cytosolic interface) to mitochondrial K ATP channels (located on the inner mitochondrial membrane) [45]. Secondly, PKC( may translocate to a different subcellular location not identified in this study. Uecker et al. identified that preconditioning promotes translocation of PKC( to sarcolemma, while PKCy redistributes to mitochondria [46]. The latter case remains to be determined for postconditioning. Thirdly, timing after ischaemia reperfusion may be critical to the levels of PKC measured in area at risk myocardium. We harvested hearts after 30 min of reperfusion, based on the fact that we detected significantly higher ppkc( levels in postconditioned hearts compared to untreated Isch/RP hearts at this time. However, if postconditioning results in transient rather than sustained translocation of PKC(, then we may have missed the critical window of opportunity in detecting an increase in PKC(. Indeed, there is some evidence to indicate that preconditioning results in transient PKC( translocation, [47] but the time course of PKC expression during postconditioning is not known. Nonetheless, our finding that selective PKC( inhibition reverses the infarct-sparing effects of postconditioning in conjunction with our observation that postconditioning results in higher ppkc( levels in total cell homogenate provides initial evidence for a role for PKC( in postconditioning cardioprotection Postconditioning s cardioprotective phenotype involves decreased translocation and phosphorylation of PKCd to mitochondria Accumulating evidence suggests that endogenous activation of PKCy during early reperfusion is responsible for initiating a deleterious signalling cascade [6,7,9,10,48 50]. Not only has PKCy been shown to rapidly translocate to mitochondria within the first 5 min of reperfusion following ischaemia [9], but also selective PKCy antagonism limits reperfusion injury [6,7,48] and associated adverse signalling events [9,10,50]. Our data corroborate the aforementioned studies by confirming that translocation of PKCy to mitochondria was observed during reperfusion and that infusion of the PKCy inhibitor, rottlerin, 5 min prior to reperfusion successfully reduced infarct size in the in vivo rat model. These data not only verify that PKCy is instrumental in mediating reperfusion injury processes, but also suggest that inhibition of PKCy induced during early reperfusion can impact upon the outcome of late reperfusion injuries. This is entirely consistent with Inagaki et al. findings that intracoronary treatment with PKCy inhibitor peptide for 1 minute only at the onset of reperfusion efficiently reduced cardiac damage induced by reperfusion in the in vivo porcine model of acute myocardial infarction [7]. Notably, this also draws attention to the possibility that postconditioning, which is also invoked during the early moments of reperfusion, may involve modulation of PKCy. To this end, postconditioning in the presence of PKCy inhibition did not result in additive infarct-sparing effects. One interpretation is that postconditioning may mediate cardioprotection via negative modulation of PKCy. Alternatively, the infarct size reduction achieved with PKCy inhibition may be maximal, therefore not providing any additional protection with postconditioning in combination with PKCy inhibition. However, this appears unlikely since remote postconditioning in the same model used in the current study confers greater protection than native postconditioning [51]. To resolve this question, we performed Western blot analysis and probed for ppkcy in cytosolic and mitochondrial fraction. Our novel data reveal that postconditioning significantly limits reperfusion-induced translocation of PKCy to mitochondria. We speculate that postconditioning may reduce ROS as a stimulus for PKCy activation. Indeed, PKCy rapidly translocates to mitochondria in hearts treated with H 2 O 2, the by-product of O 2 [9]. Observations that postconditioning significantly reduces O 2 generation and oxidant-mediated injury [44,52,53], suggest that postconditioning may inhibit oxidant-induced PKCy translocation to mitochondria Conclusion Collectively, the data presented demonstrate that the infarct-sparing effect of postconditioning is dependent on

9 A.J. Zatta et al. / Cardiovascular Research 70 (2006) PKC signalling. The protection observed appears to involve activation of PKC(, since selective isoform inhibition prevented the infarct size reduction produced by postconditioning and postconditioning was associated with significantly higher ppkc( levels in area at risk myocardium. In addition, our data reveal that postconditioning limits reperfusion-induced PKCy translocation to mitochondria. Acknowledgments This work was supported in part by NIH grant # HL (JV-J) and HL (Z-QZ), and by support from the Carlyle Fraser Heart Centre of Emory Crawford Long Hospital. Amanda J. Zatta (nee Flood) is a recipient of a Postdoctoral Fellowship from the American Heart Association. The authors wish to thank Drs. Roberta Gottlieb and Rick Sayen for their advice on isolating mitochondria, Dr. Daria Mochly-Rosen for kindly supplying the PKC( inhibitor, and Dr. Leon Chen for his valuable discussions. References [1] Vinten-Johansen J, Zhao ZQ, Zatta AJ, Kin H, Halkos ME, Kerendi F. Postconditioning a new link in nature s armor against myocardial ischemia reperfusion injury. Basic Res Cardiol 2005;100: [2] Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM. Postconditioning: a form of "Modified Reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-akt pathway. Circ Res 2004;95: [3] Yang XM, Proctor JB, Cui L, Krieg T, Downey JM, Cohen MV. Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways. J Am Coll Cardiol 2004;44: [4] Darling CE, Jiang R, Maynard M, Whittaker P, Vinten-Johansen J, Przyklenk K. FPostconditioning_ via stuttering reperfusion limits myocardial infarct size in rabbit hearts: role of ERK 1/2. Am J Physiol Heart Circ Physiol 2005;289:H [5] Yang XM, Philipp S, Downey JM, Cohen MV. Postconditioning protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3-kinase and guanylyl cyclase activation. Basic Res Cardiol 2005;100: [6] Inagaki K, Hahn HS, Dorn GW II, Mochly-Rosen D. Additive protection of the ischemic heart ex vivo by combined treatment with delta-protein kinase C inhibitor and epsilon-protein kinase C activator. Circulation 2003;108: [7] Inagaki K, Chen L, Ikeno F, Lee FH, Imahashi K, Bouley DM, et al. Inhibition of delta-protein kinase C protects against reperfusion injury of the ischemic heart in vivo. Circulation 2003;108: [8] Bright R, Raval AP, Dembner JM, Perez-Pinzon MA, Steinberg GK, Yenari MA, et al. Protein kinase C delta mediates cerebral reperfusion injury in vivo. J Neurosci 2004;24: [9] Churchill EN, Szweda LI. Translocation of delta PKC to mitochondria during cardiac reperfusion enhances superoxide anion production and induces loss in mitochondrial function. Arch Biochem Biophys 2005;439: [10] Murriel CL, Churchill E, Inagaki K, Szweda LI, Mochly-Rosen D. Protein kinase C delta activation induces apoptosis in response to cardiac ischemia and reperfusion damage: a mechanism involving BAD and the mitochondria. J Biol Chem 2004;279: [11] Liu GS, Cohen MV, Mochly-Rosen D, Downey JM. Protein kinase C- epsilon is responsible for the protection of preconditioning in rabbit cardiomyocytes. J Mol Cell Cardiol 1999;31: [12] Ludwig LM, Weihrauch D, Kersten JR, Pagel PS, Warltier DC. Protein kinase C translocation and Src protein tyrosine kinase activation mediate isoflurane-induced preconditioning in vivo: potential downstream targets of mitochondrial adenosine triphosphate-sensitive potassium channels and reactive oxygen species. Anesthesiology 2004;100: [13] Novalija E, Kevin LG, Camara AKS, Bosnjak KJ, Kampine JP, Stowe DF. Reactive oxygen species precede the epsilon isoform of protein kinase C in the anesthetic preconditioning signaling cascade. Anesthesiology 2003;99: [14] Ping P, Zhang J, Qiu Y, Tang XL, Manchikalapudi S, Cao X, et al. Ischemic preconditioning induces selective translocation of protein kinase C isoforms epsilon and eta in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res 1997;81: [15] Qiu Y, Ping P, Tang XL, Manchikalapudi S, Rizvi A, Zhang J, et al. Direct evidence that protein kinase C plays an essential role in the development of late preconditioning against myocardial stunning in conscious rabbits and that epsilon is the isoform involved. J Clin Invest 1998;101: [16] Toma O, Weber NC, Wolter JI, Obal D, Preckel B, Schlack W. Desflurane preconditioning induces time-dependent activation of protein kinase C epsilon and extracellular signal-regulated kinase 1 and 2 in the rat heart in vivo. Anesthesiology 2004;101: [17] Chiari PC, Bienengraeber MW, Pagel PS, Krolikowski JG, Kersten JR, Warltier DC. Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction: evidence for anesthetic-induced postconditioning in rabbits. Anesthesiology 2005;102: [18] Baines CP, Song CX, Zheng YT, Wang GW, Zhang J, Wang OL, et al. Protein kinase C epsilon interacts with and inhibits the permeability transition pore in cardiac mitochondria. Circ Res 2003;92: [19] Argaud L, Gateau-Roesch O, Raisky O, Loufouat J, Robert D, Ovize M. Postconditioning inhibits mitochondrial permeability transition. Circulation 2005;111: [20] Sayen MR, Gustafsson AB, Sussman MA, Molkentin JD, Gottlieb RA. Calcineurin transgenic mice have mitochondrial dysfunction and elevated superoxide production. Am J Physiol Cell Physiol 2003;284:C [21] Kin H, Zatta AJ, Lofye MT, Amerson BS, Halkos ME, Kerendi F, et al. Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine. Cardiovasc Res 2005;67: [22] Herbert JM, Augereau JM, Gleye J, Maffrand JP. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 1990;172: [23] Zhao TC, Kukreja RC. Protein kinase C-delta mediates adenosine A3 receptor-induced delayed cardioprotection in mouse. Am J Physiol Heart Circ Physiol 2003;285:H [24] Liu Y, Cohen MV, Downey JM. Chelerythrine, a highly selective protein kinase C inhibitor, blocks the anti-infarct effect of ischemic preconditioning in rabbit hearts. Cardiovasc Drugs Ther 1994;8: [25] Fryer RM, Hsu AK, Wang Y, Henry M, Eells J, Gross GJ. PKC-delta inhibition does not block preconditioning-induced preservation in mitochondrial ATP synthesis and infarct size reduction in rats. Basic Res Cardiol 2002;97: [26] Johnson JA, Gray MO, Chen CH, Mochly-Rosen D. A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function. J Biol Chem 1996;271: [27] Chen L, Wright LR, Chen CH, Oliver SF, Wender PA, Mochly-Rosen D. Molecular transporters for peptides: delivery of a cardioprotective epsilon PKC agonist peptide into cells and intact ischemic heart using a transport system, R7. Chem Biol 2001;8:

10 324 A.J. Zatta et al. / Cardiovascular Research 70 (2006) [28] Murriel CL, Mochly-Rosen D. Opposing roles of delta and epsilon PKC in cardiac ischemia and reperfusion: targeting the apoptotic machinery. Arch Biochem Biophys 2003;420: [29] Kin H, Zatta AJ, Jiang R, Reeves JG, Mykytenko J, Sorescu G, et al. Activation of opioid receptors mediates the infarct size reduction by postconditioning. J Mol Cell Cardiol 2005:38 [abstract]. [30] Cross HR, Murphy E, Bolli R, Ping P, Steenbergen C. Expression of activated PKC epsilon (PKC epsilon) protects the ischemic heart, without attenuating ischemic H+ production. J Mol Cell Cardiol 2002;34: [31] Ping P, Zhang J, Zheng YT, Li RCX, Guo Y, Bao W, et al. Cardiac targeted transgenesis of active PKC renders the heart resistant to infarction. Circulation 2000;102:II-24 [abstract]. [32] Jin ZQ, Zhou HZ, Zhu P, Honbo N, Mochly-Rosen D, Messing RO, et al. Cardioprotection mediated by sphingosine-1-phosphate and ganglioside GM-1 in wild-type and PKCepsilon knockout mouse hearts. Am J Physiol Heart Circ Physiol 2002;282:H [33] Saurin AT, Pennington DJ, Raat NJH, Latchman DS, Owen MJ, Marber MS. Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts. Cardiovasc Res 2002;55: [34] Gray MO, Zhou HZ, Schafhalter-Zoppoth I, Zhu P, Mochly-Rosen D, Messing RO. Preservation of base-line hemodynamic function and loss of inducible cardioprotection in adult mice lacking protein kinase C epsilon. J Biol Chem 2004;279: [35] Okada T, Otani H, Wu Y, Uchiyama T, Kyoi S, Hattori R, et al. Integrated pharmacological preconditioning and memory of cardioprotection: role of protein kinase C and phosphatidylinositol 3-kinase. Am J Physiol Heart Circ Physiol 2005;289:H [36] Ping P, Takano H, Zhang J, Tang XL, Qiu Y, Li RCX, et al. Isoformselective activation of protein kinase C by nitric oxide in the heart of conscious rabbits: a signaling mechanism for both nitric oxide induced and ischemia-induced preconditioning. Circ Res 1999;84: [37] Xu P, Wang J, Kodavatiganti R, Zeng Y, Kass IS. Activation of protein kinase C contributes to the isoflurane-induced improvement of functional and metabolic recovery in isolated ischemic rat hearts. Anesth Analg 2004;99: [38] Dorn GW II, Souroujon MC, Liron T, Chen CH, Gray MO, Zhou HZ, et al. Sustained in vivo cardiac protection by a rationally designed peptide that causes epsilon protein kinase C translocation. Proc Natl Acad Sci 1999;96: [39] Hausenloy DJ, Tsang A, Yellon DM. The reperfusion injury salvage kinase pathway: a common target for both ischemic preconditioning and postconditioning. Trends Cardiovasc Med 2005;15: [40] Aizawa K, Turner LA, Weihrauch D, Bosnjak KJ, Kwok WM. Protein kinase C-epsilon primes the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel to modulation by isoflurane. Anesthesiology 2004;101: [41] Obal D, Weber NC, Zacharowski K, Toma O, Dettwiler S, Wolter JI, et al. Role of protein kinase C-epsilon (PKCepsilon) in isofluraneinduced cardioprotection. Br J Anaesth 2005;94: [42] Zhang J, Bolli R, Lalli J, Tang XL, Li RCX, Zheng Y, et al. Ischemic preconditioning and phorbol ester redistribute protein kinase C epsilon to the nucleus, sarcolemmal membranes, and mitochondria in rabbit myocardium. Circulation 1999;100:I-325 [abstract]. [43] Baines CP, Zhang J, Wang GW, Zheng YT, Xiu JX, Cardwell EM, et al. Mitochondrial PKCepsilon and MAPK form signaling modules in the murine heart: enhanced mitochondrial PKCepsilon MAPK interactions and differential MAPK activation in PKCepsilon-induced cardioprotection. Circ Res 2002;90: [44] Sun HY, Wang NP, Kerendi F, Halkos M, Kin H, Guyton RA, et al. Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca 2+ overload. Am J Physiol Heart Circ Physiol 2005;288:H [45] Costa ADT, Garlid KD, West IC, Lincoln TM, Downey JM, Cohen MV, et al. Protein kinase G transmits the cardioprotective signal from cytosol to mitochondria. Circ Res 2005;97: [46] Uecker M, da Silva R, Grampp T, Pasch T, Schaub MC, Zaugg M. Translocation of protein kinase C isoforms to subcellular targets in ischemic and anesthetic preconditioning. Anesthesiology 2003;99: [47] Simonis G, Weinbrenner C, Strasser RH. Ischemic preconditioning promotes a transient, but not sustained translocation of protein kinase C and sensitization of adenylyl cyclase. Basic Res Cardiol 2003;98: [48] Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, et al. Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC. Proc Natl Acad Sci 2001;98: [49] Chou WH, Choi DS, Zhang H, Mu D, McMahon T, Kharazia VN, et al. Neutrophil protein kinase delta as a mediator of stroke-reperfusion injury. J Clin Invest 2004;114: [50] Churchill EN, Murriel CL, Chen CH, Mochly-Rosen D, Szweda LI. Reperfusion-induced translocation of deltapkc to cardiac mitochondria prevents pyruvate dehydrogenase reactivation. Circ Res 2005; 97: [51] Kerendi F, Kin H, Halkos ME, Jiang R, Zatta AJ, Zhao ZG, et al. Remote postconditioning: brief renal ischemia and reperfusion applied before coronary artery reperfusion reduces myocardial infarct size via endogenous activation of adenosine receptors. Basic Res Cardiol 2005;100: [52] Halkos ME, Kerendi F, Corvera JS, Wang NP, Kin H, Payne CS, et al. Myocardial protection with postconditioning is not enhanced by ischemic preconditioning. Ann Thorac Surg 2004;78: [53] Kin H, Zhao ZQ, Sun HY, Wang NP, Corvera JS, Halkos ME, et al. Postconditioning attenuates myocardial ischemia reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res 2004;62:74 85.